Purification and Characterization of a Novel Type I Ribosome Inactivating Protein, Pachyerosin, from Pachyrhizus erosus Seeds, and Preparation of its Immunotoxin against Human Hepatoma Cells

Abstract

Pachyrhizus erosus seeds have a high protein content and are used in China due to their cytotoxic effect. Here we report the biological and pharmacological activity of the protein extracts from P. erosus seeds. A novel ribosome-inactivating protein, pachyerosin, from P. erosus seeds was successively purified to homogeneity using ammonium sulfate precipitation, DEAE-sepharose FF, and Sephacryl S-200. Pachyerosin showed to be a type I ribosome-inactivating protein with a molecular mass of 29 kDa and an isoelectric point of 9.19. It strongly inhibited protein synthesis of rabbit reticulocyte lysate with an IC₅₀ of 0.37 ng/mL and showed N-glycosidase activity on rat liver ribosomes with an EC₅₀ of 85.9 pM. The N-terminal 27 amino acids of pachyerosin revealed a 60.71 % sequence identity with abrin A from the seeds of Abrus precatorius. With the aim of targeting the delivery of pachyerosin, immunotoxin was prepared by conjugating pachyerosin with anti-human AFP monoclonal antibodies SM0736. The immunotoxin pachyerosin-SM0736 efficiently inhibited the growth of the human hepatoma cell line HuH-7 with an IC₅₀ of 0.050 ± 0.004 nM, 2360 times lower than that of pachyerosin and 430 times lower than that of the immunotoxin against human gastric cancer cell line SGC7901. These results imply that pachyerosin may be used as a new promising anticancer agent.

Key words

Pachyrhizus erosus - Fabaceae - purification - ribosome-inactivating proteins - immunotoxin - anticancer agent

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